The irregular xylem3 (irx3) mutant of Arabidopsis has a severe deficiency in secondary cell wall cellulose deposition that leads to collapsed xylem cells. The irx3 mutation has been mapped to the top arm of chromosome V near the marker nga106. Expressed sequence tag clone 75G11, which exhibits sequence similarity to cellulose synthase, was found to be tightly linked to irx3, and genomic clones containing the gene corresponding to clone 75G11 complemented the irx3 mutation. Thus, the IRX3 gene encodes a cellulose synthase component that is specifically required for the synthesis of cellulose in the secondary cell wall. The irx3 mutant allele contains a stop codon that truncates the gene product by 168 amino acids, suggesting that this allele is null. Furthermore, in contrast to radial swelling1 (rsw1) plants, irx3 plants show no increase in the accumulation of beta-1,4-linked glucose in the noncrystalline cell wall fraction. IRX3 and RSW1 fall into a distinct subgroup (Csa) of Arabidopsis genes showing homology to bacterial cellulose synthases.